The primary role of acetylcholinesterase (AChE) is the termination of impulse transmission in cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine (ACh). Some organophosphate (OP) compounds, such as the nerve agent sarin and soman, or agricultural pesticides, inhibit cholinesterases (ChEs), such as acetylcholinesterases (AChEs) and butyrylcholinesterases (BChEs), by rapid phosphylation of the serine residue in the enzyme active site. The acute toxicity of these OP agents leads to motor and respiratory failure due to the inhibition of AChE in the peripheral and central nervous system.
The high reactivity of ChEs towards OP-agents led to propose these biomolecules, as exogenous scavengers for sequestration of toxic OP-agents, or as destroyers, before they reach their physiological target. However, a large-scale production of suitable serine hydrolases with OP scavenging and hydrolytic activity is obstructed by the fact that recombinant enzymes of various origins are relatively short-lived in the circulation of experimental animals [e.g., Kronman C. et al.: Biochem. J. 311 (1995) 959-67; Chitlaru T. et al.: Biochem. J. 336 (1998) 647-58]. In a controlled conjugation of polyethylene glycol (PEG) moieties to the C-terminal truncated version of recombinant human AChE (ΔC-rHuAChE), we increased the life-time of the enzyme activity in the circulation of mice (WO 02/087624). Examination of pharmacokinetic performance of an array of PEGylated (=pegylated) AChEs which differed one from another by their degree of modification, as well as by the length of the appended PEG chains, demonstrated that circulatory residence of the enzyme was significantly improved, and that the mean residual time (MRT) was increased as high as 50-fold when compared to the MRT of nonmodified rHuAChE. MRT, reflecting the average length of time of retaining the administered molecules by the organism, is obtained by analyzing the clearance data according to a non-compartmental pharmacokinetic model, as opposed to the half-life value, which is derived by fitting the clearance profile to a bi-exponential pharmacokinetic model. Said modified HuAChE exhibited MRT values of 2100 minutes and more in mice. Such values are unmatched by any other known form of recombinant or native plasma derived cholinesterase (ChE) reported to date in mice. The maximal MRT values reported in mice for serum derived human BChE, horse BChE, or fetal bovine AChE, have been approximately 1,400 minutes [e.g., Kronman C. et al.: Biochem. J. 311 1995) 959-67]. We have further demonstrated the increase of circulatory residence of pegylated rHuAChE in rhesus macaques [Cohen O. et al.: Biochem. J. 378 (2004) 117-28)].
The substantial increase in the pharmacological stability of ChEs, mentioned above, has removed an obstacle on the way toward utilizing serine hydrolases as organophosphates scavengers. However, an enzyme-based detoxifying agent for pharmaceutical uses should exhibit, beside improved stability and retained activity, preferably also a well defined composition. A desired agent for a pharmaceutical use should preferably comprise one chemical structure, but pegylation of a protein provides a heterogeneous product corresponding to a mixture of various structures comprising randomly formed linkages of PEG with reactive sites, such as lysine 1-amino groups, that are available on the protein molecule. If, for example, a protein has 8 free amino acids, similarly accessible for coupling with PEG molecules, theoretically 256 different combinations may be obtained. Although in practical cases not all possibilities will be realized with the same probability, a heterogeneous composition will be obtained, macroscopically appearing usually as more components. It is therefore an object of this invention to provide a uniformly pegylated preparation of serine hydrolase.
US Patent Application 2004/0082765 describes erythropoietin conjugated with one PEG chain at one position only, taking advantage of low number of available amino groups in the protein. However, in general cases there are many free attachment sites available, and furthermore, it is usually preferable to couple more than one polymer chain to the protein. It is therefore another object of this invention to provide a general method for the preparation of uniformly pegylated serine hydrolases comprising any predetermined number and any predetermined locations of the attachment sites.
Onda et al. [Onda M. et al.: Bioconjugate Chem. 14 (2003) 480-7] described the preparation of mutants of a recombinant immunotoxin with diminished number of lysine residues for eventual pegylation, and assessed the effect of the mutations on the residual immunotoxin activity. US Patent Application 2005/0114037 relates to a computational method predicting how the location and type of coupling might decrease specific activity of said protein. This invention is directed predominantly to decreasing immunogenicity of a conjugated protein. It is also an object of the invention to provide a method for the preparation of uniformly pegylated serine hydrolase with organophosphate scavenging or hydrolytic activity with lowered immunogenicity, comprising steps of predetermining the number and the location of the conjugated PEG chains, possibly utilizing three-dimensional structures of mutants lacking certain free amino groups present in the wild type, and selecting suitable mutants according to accessibility of the free amino groups for eventual coupling.
It is still another object of this invention to provide a homogeneous preparation of pegylated serine hydrolase exhibiting extended circulatory residence time in the mammalian circulation.
It is a further object of this invention to provide a uniformly pegylated preparation of acetylcholinesterase displaying mean residence time (MRT) values of 1000 minutes or more in the mouse circulation or 4000 minutes or more in the circulation of primates.
It is a still further object of this invention to provide a method for producing uniformly pegylated AChE, exhibiting sufficient activity and circulatory longevity in the circulation of primates.
It is also another object of this invention to provide a homogeneous agent based on pegylated AChE for use in scavenging organophosphates (OPs).
This invention also aims at providing the use of pegylated AChE in preventive and therapeutic treatment of OPs poisoning.
It is also an object of this invention to provide a pharmaceutical composition for treating or preventing OPs poisoning, which composition exhibits low immunogenicity.
Other objects and advantages of present invention will appear as description proceeds.